The wide-ranging effort to sequence the human genome has generated new sub-fields of research and new research challenges. The sequencing effort identifies new genes which encode heretofore unknown proteins. Efforts at characterizing the proteins focus on the important issue of determining what the protein does in the body and in what tissues, cells, or subcellular organelles the protein is found. Thus, with the human genome project proceeding at full steam, there is increased need for research methods to characterize new proteins.
The protein-encoding genes, which are made of two strands of material called deoxyribonucleic acid (DNA), encode information used to construct proteins via an intermediate which is a molecule of ribonucleic acid (RNA). The process of transcribing the information encoded in one strand of the DNA of a gene into a complementary molecule, the RNA, is called "transcription" and the resulting RNA strand is referred to as a "transcript" or as "messenger RNA" (mRNA). "Translation" refers to the process by which the information encoded in the mRNA transcript is used, as a blueprint, to synthesize a polypeptide chain. One or more polypeptide chains compose a protein.
Following synthesis of the polypeptide chain, the chain folds into its unique conformation that is characteristic for each protein and may undergo further modifications by processes known as "post-translational processing." If these modifications occur as the polypeptide is being synthesized, then the term "co-translational processing" is used. Another aspect to the maturation of a polypeptide chain into a mature protein is "translocation," the movement of the polypeptide chain across a biological membrane. Some aspects of translational processing occur contemporaneously with translocation and, in talking about these processing events, the term "co-translational translocation" is used.
To study the general phenomenon of protein synthesis and processing in the laboratory, cell-free systems are used. There are a number of known cell-free systems for translation, which are prepared from a variety of sources, including E. coli, wheat germ, Xenopus eggs, rabbit reticulocyte, and HepG2 cells (Frydman, J. and Hartl, F U, (1996) Science 272:1497-1502; Goldstein, B. J. and Kahn, C. R. (1988) J. Biol. Chem. 263:12809-12). Two widely used in vitro translation systems are rabbit reticulocyte lysate (RRL) and wheat germ extract. The RRL and wheat germ extract systems are commercially available as translation kits and are relatively straightforward to use. These kits allow for translation of a nascent polypeptide.
In cells, the biosynthesis of many proteins requires co-translational translocation across membranes of an organelle called the endoplasmic reticulum (ER) for proper processing. In cell-free systems, in place of the ER, microsomal membranes are used, which are equivalent to the ER in that they contain a high percentage of ER membrane which have been isolated by centrifugation.
Cell-free translation systems such as RRL contain very few ER membrane equivalent, so that in order to observe co-translational translocation and core glycosylation processing, an exogenous source of microsomes is needed (Zhou, X., Baker, N. K., and Arakaki, R. F. (1993) Biochem. Biophys. Res. Comm. 192:1453-1459). Examination of co- and post-translational protein modifications of polypeptides has been performed by the addition of heterogeneous canine pancreatic microsome membranes (MM). Canine pancreatic microsomal membranes have been used in combination with RRL to study the post-translational processing of both secretory proteins and membrane proteins, but the processing efficiency for membrane proteins is limited. Since a large proportion of proteins are either secretory or membrane-bound proteins, and since biosynthesis of these proteins requires serial posttranslational modifications initiated in the ER, the use of efficient cell-free translation and translocation methods is needed.
The RRL system was used to study the structure and functional characteristics of the human insulin receptor protein (hINSR) during its biosynthesis (Zhou, X., Baker, N. K., and Arakaki, R. F. (1993) Biochem. Biophys. Res. Comm. 192:1453-1459). Processing of the hINSR precursor protein by the addition of microsomal membranes was of consistently low efficiency. This may reflect the fact that the hINSR precursor is a large transmembrane protein (i.e., it spans the entire width of a biological membrane in which it is embedded) of about 160 kDa, and processing by microsomal membranes requires multiple and extensive N-linked glycosylation steps to yield the 190 kDa pro-receptor. Large transmembrane proteins, such as hINSR, may exceed the capacity of canine microsomal membranes to efficiently process the protein synthesized in a RRL system.
A further problem with canine microsomal membranes, is that the low efficiency of protein processings requires that additional steps be performed, such as lectin chromatography to separate and isolate the 190 kDa pro-receptor from the 160 kDa nascent protein receptor precursor.